Keratinocytes and dermal endothelial cells are the main source of soluble factors regulating healing of skin wounds and ulcers. Abnormalities due to decreased activities including growth factor production, angiogenic response, macrophage function, collagen accumulation, epidermal barrier function, and keratinocyte and fibroblast migration and proliferation, all contribute to defective wound healing. Growth factors and cytokines have been used in clinical settings for treating wounds. Examples include, but not limited to, PDGF (platelet-derived growth factor (Rees et al, 1999) and GM-CSF (granulocyte-macrophage colony stimulating factor) that have been shown to exert beneficial effects on wound healing in patients suffering from various wounds and chronic skin ulcers of diverse etiology including hydroxyurea-related leg ulcers (Stagno et al, 1999), venous leg ulcers (Da Costa et al, 1999), hemoglobinopathy-related ulcers (Voskaridou et al, 1999), and wounds resulting from amputation (Gaches et al, 1998). Also intradermal administration of GM-CSF to leprosy patients with skin lesions leads to enhanced wound healing and increased numbers and layers of keratinocytes (Kaplan et al, 1992; Braunstein et al, 1994).
Both growth factors and cytokines are proteins. Difficulties with the use of protein therapeutics to treat epidermal wounds are often related to the large size of the proteins involved. The complex structure and the cost of manufacture of natural proteins are prohibitive for wide clinical use. Stability and compatibility of such natural proteins in formulation are also a major concern. The poor penetration due to large size of natural proteins to reach the target layer of skin often reduce the efficacy and account for failed beneficial effects of protein therapeutics. To overcome these issues, short peptides that bear the activity of large proteins should fill the need of less expensive, cost effective production, and simple handling and manipulation. In addition, short bioactive peptides are better absorbed and retained by wound tissue due to less susceptibility to protease. The advantage of absorption characteristics of short bioactive peptides also make them viable option for uses beyond the care of acute and chronic wounds, such as for treatment of the skin problems associated with aging and sun exposure.
Chemokines are structurally related and represent a large superfamily of 8 to 150 kd proteins that possess diverse biological activities. They are usually secreted upon cell stimulation to control leukocytes trafficking during homeostasis, as well as during inflammation, and are necessary for the linkage between innate and adaptive immunity. Along with adhesion molecules such as integrins and selectins, chemokines and their receptors act primarily as part of a complex molecular network that facilitates the selective movement of specific cell types into, and out of, targeted tissue microenvironment (Key et al., 2003; Ono et al., 2003). Chemokines selectively mediate the regionally specific recruitment of neutrophils, macrophages and lymphocytes. In addition to being chemotactic factors, the chemokines also play important roles in maintenance of homeostasis, angiogenesis/angiostasis, cellular differentiation and activation, wound healing, tumor growth and metastasis, lymphocyte homing and development of lymphoid tissue, and influencing the overall type 1/type 2 balance of immune response (Behm et al., 2012; Gillitzer et al., 2001; Raman et al., 2011; Romagnani et al., 2004; Rossi et al., 2000).
Defined by a tetra cysteine motif, the chemokines are subdivided into four distinct families according to the configurations of the cysteine residues at their amino terminus. There are two large subfamilies, CCL subfamily (CCL1 through CCL28) and CXCL subfamily (CXCL1 through CXCL16), as well as two small subfamilies, XCL subfamily (XCL1 through XCL2) and CX3CL1 subfamily (Bacon et al. 2003). The CXC subfamily of chemokines plays an important role in diverse processes, including inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis (Keeley et al., 2008; 2011). Many chemokines interact with the glycosaminoglycan (GAG) moieties of proteoglycans on endothelial cells and the extracellular matrix (Handel et al., 2005). Heparin, which serves as a model compound for heparin sulfate, is the most ubiquitous class of GAG that is expressed on virtually every cell in the body. All chemokines interact with heparin GAG.
In our studies we noted the C-X-C chemokines showed some sequence similarities in their primary amino acid sequences although highly conserved in the secondary structures. Examination of the primary amino acid sequences of nine human C-X-C chemokines reveals a highly conserved region located at the C-terminal portion which is involved in GAG binding using the NCBI accession number of each chemokines shown below in “Detailed description of the invention, 1st paragraph”. We generated tetrapeptides from the GAG binding region and tested the bioactivity. To our surprise the tetrapeptides showed diverse bioactivities including promote keratinocyte migration, induce angiogenesis on human umbilical vein endothelial cells, neutralize LTA induced pro-inflammatory cytokines, and modulating cell growth and growth factor production. The tetrapeptides are useful as both pharmaceutical and cosmetic products for improving various skin conditions.